Brain Proteomics in Niemann-Pick Disease- Type C (NPC). We have characterized cerebellar tissue from five weeks old littermate control (n=7) and Npc1 mutant female mice (Npc1m1N, n=7). After statistical analysis of 871 protein spots on quadruplicate gels, a total of 52 protein spots were found to be significantly increased (R&#8805;1.20, p<0.05) and 12 protein spots significantly decreased (R&#8804;0.83, p<0.05) in Npc1-/- cerebellar tissue. Differentially expressed protein spots were isolated from paired mutant and control preparative gels, and using mass spectrometric techniques we have candidate identifications, to date, on 20 pairs of protein spots. Although validation is pending, one of the identified proteins is transthyretin. Although this protein identification was made in mouse brain, transthyretin is an intriguing potential biomarker in human CSF. One reason for this is that decreased levels of cerebral spinal fluid transthyretin correlate with disease severity in Alzheimer disease. These studies have now been extended to one and three weeks old littermates and a total of 200 additional differentially expressed gel spots. Cerebral Spinal Fluid Profiling (CSF) in NPC. Initial experiments have demonstrated that we can detect significant differences between cerebral spinal fluid from NPC patients and adult controls using our novel analytical approach to compare protein profiles. CSF samples have been collected from 37 well-characterized NPC patients. Currently the collection for NPC patients includes longitudinal samples collected over 2.5 years and five pre/post miglustat pairs. In addition a collection of pediatric CSF samples is in progress at Childrens National Medical Center. Our analytical approach accounts for systemic sources of variance, employs an analysis of variance (ANOVA) approach to the sets of MALDI-TOF spectra, and then applies Principal Component Analysis to the data arrays generated by ANOVA. The results of ANOVA-PCA analyses will be correlated with patient disease status using a neurological severity scale that can be used to define disease status and progression in NPC patients as developed by the SMD, NICHD. The extensive differentially observed profile spectra are being analyzed further using mathematical tools of Fuzzy multivariate rule-building expert systemsminimal neural networks (FuRES) to elucidate potential specific biomarkers. Initial results of this analysis show the presence of at least 10 specific peptide masses that are candidate biomarkers. We will employ chromatographic separation of the CSF followed by mass spectrometry to identify specific peptides in the CSF suggested by the 95% Confidence Intervals of the FuRES analysis. Lipid Quantification in Serum. We have continued developing methodology to quantify cardiolipins in human serum by mass spectrometry. This effort is in association with a clinical study underway in the Institute to evaluate the effects of antibiotic treatment of pregnant women colonized with the Group B streptococcal (GBS) organism. The hypothesis of the study is that the typical peri-natal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant which then leads to respiratory distress. It has been demonstrated in other studies in newborn sheep that the GBS organisms secrete a specific cell wall membrane cardiolipin with penicillin treatment and that this substance causes respiratory distress at levels corresponding to the injection of about 100 pmole/mL in serum;note that this concentration falls rapidly with a half life of a few minutes. It is not known whether the respiratory distress observed in a fraction of infants born to GBS colonized mothers is a result of a similar effect, or perhaps by a related effect caused by a release of endogenous cardiolipins stimulated by the bacterial death. The analytical approach involves the addition of an internal standard to a serum sample, extractions by a combination of liquid-liquid and solid phase, followed by an LC-MS analysis that incorporates an extraction/recovery standard to monitor system quality control. We have shown that cardiolipin can be extracted from serum with approximately 90% efficiency. We have further shown that normal adult levels of (18:2)4 cardiolipins are present in serum at levels <10 fmole/uL, approximately 1000-fold lower than found by earlier, less accurate measurements. With this base line information, we are seeking to establish normal serum levels in cord blood of mothers known to be uncolonized by GBS and will then determine levels in infected individuals.